This invention generally relates to systems that use automated image processing techniques to detect and quantify agglutinates formed in response to immunological agglutination reactions. More particularly, the present invention relates to methods and apparatus for calibrating such systems.
Immunological agglutination reactions are used to identify blood types and to detect antibodies and antigens in blood samples and other aqueous solutions. In a conventional procedure, a sample of red blood cells is mixed with serum or plasma in test tubes or micro plates, and the mixture is then centrifuged. Commonly, but not necessarily, the mixture is also incubated before being centrifuged. Various reactions either occur or do not occur in the mixture depending on, for example, the blood type of the red blood cells, or whether certain antibodies are present in the blood sample.
Typically, these reactions manifest themselves as clumps of cells or particles, referred to as agglutinates, having antigens and antibodies on their surfaces. The absence of any such clumps thus indicates that no reaction has occurred, and the presence of such clumps indicates that a reaction has occurred. In addition, if a reaction has occurred, then the size and amount of the formed clumps are quantitative indicators of the level or concentration in the sample of the complex for which the blood sample was tested. The size and amount of the formed clumps are also quantitative indicators of the affinity of that complex for the reagent used to produce the reaction.
Recently, a new agglutination test method--referred to as column agglutination technology, or CAT--has been developed. Column agglutination technology may be defined as the analysis of blood and blood products utilizing filtration as a means of separating agglutinated, precipitated, absorbed, or adsorbed particulate components from non reactive components for immunoassay applications. In this method, gel or glass bead micro particles are contained within a small column, referred to as a microcolumn. A reagent such as anti-IgG is dispensed in a diluent in the microcolumn and a test red blood sample is placed in a reaction chamber above the column. The column, which is typically one of a multitude of columns formed in a transparent cassette, is then centrifuged.
The centrifuging accelerates the reaction, if any, between the reagent and the blood sample and also urges the cells of the blood sample toward the bottom of the column. The glass beads or gel in the microcolumn act as a filter, however, and resist or impede downward movement of the particles in the column. As a result, the nature and distribution of the particles in the microcolumn after centrifuging provide a visual indication of whether any agglutination reaction occurred in the microcolumn, and if so, of the strength of that reaction.
In particular, if no agglutination reaction occurs, then all or virtually all of the cells of the blood sample in the microcolumn pass downward during centrifuging to the bottom of the column, and these cells form a pellet at that bottom. In contrast, if there is a very strong reaction between the reagent and the blood sample, virtually all of the cells of the sample agglutinate, and large agglutinates form at the top of the microcolumn, above the gel or glass beads contained therein. The gel or glass beads prevent the agglutinates from passing to the bottom of the column during centrifuging, so that after centrifuging, the agglutinates remain above the gel or beads.
If there is a reaction between the reagent and the blood sample, but this reaction is not as strong as the above-described very strong reaction, then some but not all of the cells of the blood sample agglutinate. The percentage of the cells that agglutinate and the size of the agglutinated particles both vary directly with the strength of the reaction.
During centrifuging, the unreacted cells pass to the bottom of the column, and the distance that the agglutinated particles pass downward through the column depends on the size and number of the particles. Hence, the size of the pellet of cells at the bottom of the microcolumn, and the extent to which the agglutinates penetrate into the gel or glass beads in the microcolumn, are both inversely related to the strength of the reaction between the reagent and the blood sample.
Conventionally, an agglutination reaction pattern is classified as either negative or positive, and if positive, the reaction is then further classified into one of a series of classes depending on the strength of the reaction. Traditionally, the classification is done by a human technician or operator who observes, or reads, the reaction pattern in the column. The use of human technicians for this purpose has several disadvantages. For example, the technicians need to be highly skilled and trained to read and to classify the reactions properly. Also, even with highly skilled and trained technicians, the classifications are subject to human interpretation, and as a result, it is believed that the consistency and reproducibility of the classifications can be improved. Because of these disadvantages, efforts have been made to automate the classification of the agglutination reactions.
One automated system for reading and classifying agglutination reactions in microcolumns is disclosed in copending application Ser. No. 08/163,996 for "Method and System For Classifying Agglutination Reactions." The method disclosed in this copending application is based on a computerized imaging system.
In accordance with this method, an image of an agglutination reaction is formed on an array of pixels, and those pixels generate electric charges that are converted to digital data values. These data values are then processed according to a predetermined program to determine if an agglutination pattern is present in the image, and, if so, to classify that pattern into one of a plurality of predefined classes.
In order to obtain consistent test results with this system, it is desirable to calibrate the system regularly.